J Korean Soc Endocrinol.  2006 Jun;21(3):192-203. 10.3803/jkes.2006.21.3.192.

Retrovirus Mediated Gene Transfer of RANK-Fc Ameliorates Bone Loss in a Mouse Ovariectomy Model of Osteoporosis

• Kim D
• Shin CS

• KMID: 2200731
• DOI: http://doi.org/10.3803/jkes.2006.21.3.192

Abstract

BACKGROUND: Interactions between the receptor activator of the NF-kappaB ligand (RANKL) and its receptor, RANK, are important in the terminal differentiation and activation of osteoclasts. In the current investigation, we examine the feasibility of using genetically modified mesenchymal stem cells (MSCs), C3H10T1/2 cells as a platform for the sustained systemic delivery of therapeutic proteins into the circulation in an osteoporosis model, and investigate retroviral-mediated gene therapy of RANK-Fc as a means of ameliorating ovariectomy (OVX)-induced bone resorption.
METHODS
C3H10T1/2 cells were transduced with a MSCV-based retroviral vector containing cDNA of a fusion protein combining the extracellular domain of murine RANK with the human immunoglobulin constant domain (MSCV-RANK-Fc-eGFP). Young adult female mice were subjected to OVX or sham surgery, followed by treatment with transduced cells or PBS 4 weeks later. The expression of RANK-Fc by these cells was assessed, both in vitro and in vivo. Total bone mineral density (BMD) was measured and GFP expression was examined.
RESULTS
Transduced cells produced biologically active RANK-Fc in vitro and in vivo. Mice that were subjected to OVX followed by treatment with cells transduced with MSCV-RANK-Fc-eGFP 4 weeks later contained no significant but higher total BMD than either the control vector or PBS-treated mice after 8 weeks. Higher GFP expression was attained in the liver, spleen, and intra-abdominal fat of mice treated with MSCV-RANK-Fc-eGFP.
CONCLUSION
The data collectively indicate that C3H10T1/2 cells are effectively transduced with a MSCV-based retrovirus, and are capable of secreting biologically active RANK-Fc in vitro and in vivo. Moreover, gene therapy facilitating the sustained delivery of RANK-Fc may be an effective method to reverse OVX-induced osteoporosis.

MeSH Terms

Animals
Bone Density
Bone Resorption
DNA, Complementary
Female
Genetic Therapy
Humans
Immunoglobulins
Intra-Abdominal Fat
Liver
Mesenchymal Stromal Cells
Mice*
NF-kappa B
Osteoclasts
Osteoporosis*
Ovariectomy*
Retroviridae*
Spleen
Young Adult
Zidovudine
DNA, Complementary
Immunoglobulins
NF-kappa B
Zidovudine

Figure

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  Fig. 1 Schematic representation of retroviral vectors. RANK-Fc cDNA is located downstream of MSCV LTR and upstream of IRES-eGFP cassette, allowing the bicistronic expression of both RANK-Fc and eGFP.

• 

  Fig. 2 Expression of GFP in transduced C3H10T1/2 cells. The left panels depict light microscopy images, and right panels show images at the same position under fluorescent light. Cells transduced with MSCV-RANK-Fc-eGFP (A) and MSCV-eGFP (B) express GFP (98.3% and 85.3%, respectively) (magnification ×100).

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  Fig. 3 Cellular expression of RANK-Fc in vitro. Western blot analysis of cellular lysates from transduced Chinese hamster ovarian (CHO) cells. Blots were probed with goat polyclonal anti-mouse RANK antibody (A) and goat polyclonal anti-human IgG Fc fragment-specific antibody (B). Lane 1, pMSCV-eGFP-transduced CHO cells; lane 2, pMSCV-RANK-Fc-eGFP-transduced CHO cells. The numbers on the both sides indicate molecular size markers in kDa.

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  Fig. 4 Western blot analysis of RANK-Fc secreted from C3H10T1/2 cells transduced in vitro. Protein samples from conditioned medium were immunoprecipitated with goat polyclonal anti-mouse RANK antibody and resolved by SDS-PAGE. Blots were probed with goat polyclonal anti-mouse RANK antibody (A) and goat polyclonal anti-human IgG Fc fragment-specific antibody (B). Lane 1, MSCV-RANK-Fc-eGFP-transduced C3H10T1/2 cells; Lane 2, MSCV-eGFP-transduced C3H10T1/2 cells. The numbers on the right signify molecular size markers in kDa.

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  Fig. 5 Systemic physiologic changes observed at 28 days after OVX. Six week-old female C3H mice were sham operated [closed square, n = 4] or ovariectomized [open square, n = 14], and bone mineral density (BMD) (A), bone mineral content (BMC) (B), body weight (C), and body fat mass (D) were measured at baseline and four weeks after the operation. Data are expressed as means and standard deviations. *P < 0.05, OVX group vs. sham op group (non-parametric Mann-Whitney test). †P < 0.01, OVX group vs. sham op group (non-parametric Mann-Whitney test).

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  Fig. 6 RANK-Fc prevents the progression of established osteopenia in C3H mice. A, Schematic representation of the experimental protocol; B, The effect of RANK-Fc on bone mineral density over time. Sham op (closed square, n = 2); OVX + Vehicle (open square, n = 3); OVX +GFP (open circle, n = 2); OVX + RANK-Fc (closed circle, n = 4); C, Changes in bone mineral density after injection; D, Changes in bone mineral density, compared to the baseline. Sham op (open bar, n = 2); OVX + Vehicle (gray bar, n = 3); OVX + GFP (slashed bar, n = 2); OVX + RANK-Fc (black bar, n = 4). Data are expressed as means and standard deviations. *P < 0.05, OVX + RANK-Fc vs. OVX + Vehicle (non-parametric Kruskal-Wallis test).

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  Fig. 7 Whole-body radiographs in C3H mice. C3H mice were injected i.p. with transduced C3H10T1/2 cells or PBS at 4 weeks after OVX or sham op. No significant differences in radiodensity were evident after 8 weeks.

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  Fig. 8 Production of RANK-Fc in mice. C3H10T1/2 cells were transduced with MSCV-RANK-Fc-eGFP and MSCV-eGFP. Eleven week-old C3H mice were administered transduced cells or PBS on Days 0, 2, 4, and 6 at 4 weeks after OVX. Serum RANK-Fc levels were measured by ELISA on Days 14, 28, and 56 after the first injection. Data are expressed as means and standard deviations (n = 2-4/group).

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  Fig. 9 Localization of RANK-Fc-secreting cells in tissue sections of transduced C3H10T1/2 cell transplants C3H10T1/2 cells were infected with MSCV-RANK-Fc-eGFP in vitro, and transplanted into ovariectomized C3H mice. After 8 weeks in vivo, transplants were harvested and prepared for the histological evaluation of GFP expression. Higher expression of GFP was attained in liver (A), spleen (B), and intra-abdominal fat (C) in "OVX + RANK-Fc" mice (right), compared with that in corresponding tissues from "sham op" mice (left) (magnification ×100).

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  Fig. 10 Localization of RANK-Fc-secreting cells in bone and bone marrow of transplants of transduced C3H10T1/2 cells. C3H10T1/2 cells were infected with MSCV-RANK-Fc-eGFP in vitro, and transplanted into ovariectomized mice. After 8 weeks in vivo, femurs were harvested and prepared for histological evaluation of GFP expression (A), or freshly isolated cell culture from bone marrow was obtained (B) No GFP signal was detected in the "sham op" (left) and "OVX + RANK-Fc" (right) groups (magnification ×100).

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